Technical Abstract:
Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel transcript which is represented by multiple ESTs derived only from the oocyte cDNA library. Analysis of tissue distribution of the novel transcript by reverse transcription polymerase chain reaction revealed that the novel gene is only expressed in unfertilized oocytes but not in 10 somatic tissues examined. Northern blot analysis showed the transcript size is approximately 2 kb. The full-length cDNA sequence for the novel gene was obtained by assembly of sequences from the EST clones and a 5'end sequence from a 5'RACE product. The complete cDNA is 1966 bp in length containing an open reading frame which encodes a predicted protein of 514 amino acids. The amino acid sequence does not share homology with any known proteins in the NCBI databases. However, a search of the Pfam protein database revealed that the protein contains an F-box motif at the N-terminus, indicating that this novel oocyte-specific protein is a member of the rapidly expanding family of F-box proteins. F-box proteins are components of ubiquitin-ligase complexes, in which they bind substrates for uiquitin-mediated proteolysis. F-box proteins have also been associated with cellular functions such as signal transduction and regulation of the cell cycle. Real time PCR analysis of the mRNA expression of the novel gene during vitellogenesis and early embryogenesis demonstrated that the expression of the novel gene is extremely high during early previtellogensis and decreases dramatically after fertilization. This result is confirmed by in situ hybridization which showed that the novel transcript is highly abundant in early previtellogenic oocytes. Western blot analysis using antibodies generated against a synthetic peptide of the novel protein detected a protein band of ~51 kDa in a protein sample prepared from oocytes. The specific expression of this gene in oocyte and its developmental pattern of expression suggest a role for this novel gene in the regulation of early development of oocyte.